Biomaterial Database

Dihydroorotase from Escherichia coli. Substitution of Co(II) for the active site Zn(II). 1991

D C Brown, and K D Collins

Department of Biological Chemistry, University of Maryland Medical School, Baltimore 21201.

Treatment of Escherichia coli dihydroorotase (a homodimer of subunit molecular weight 38,729) containing only the 1 active site Zn(II) ion per subunit with the sulfhydryl reagent N-(ethyl)-maleimide (NEM) blocks the two external Zn(II) sites per subunit and dramatically lessens the precipitation caused by high concentrations of Zn(II); stabilizes the enzyme partially against air oxidation and dilution inactivation; makes the active site Zn(II) easier to remove; and lowers Km and increases kcat. Treatment of NEM-blocked dihydroorotase ((NEM)dihydroorotase) with the chelator 2,6-pyridinedicarboxylic acid at pH 5.0 in the absence of oxygen and trace metal ions removes the active site Zn(II) with a half-life of 15 min, allowing the production of milligram amounts of moderately stable apo-(NEM)dihydroorotase in about 80% yield. Treatment of apo-(NEM)dihydroorotase with Co(II) at pH 7.0 produces (NEM)dihydroorotase completely substituted at the active site with Co(II) in 100% yield: analysis gives 0.95-1.1 g atoms of Co(II) per active site and 0.03-0.05 g atoms of Zn(II) per active site. This Co(II)-(NEM)dihydroorotase is hyperactive at pH 8. The electronic absorption spectrum of Co(II)-(NEM)dihydroorotase at pH 6.5 implicates an active site thiol group as a ligand to the metal ion. The spectrum is inconsistent with tetrahedral coordination of the active site metal ion and is most consistent with a pentacoordinate structure.

UI	MeSH Term	Description	Entries
D007700	Kinetics	The rate dynamics in chemical or physical systems.
D008345	Manganese	A trace element with atomic symbol Mn, atomic number 25, and atomic weight 54.94. It is concentrated in cell mitochondria, mostly in the pituitary gland, liver, pancreas, kidney, and bone, influences the synthesis of mucopolysaccharides, stimulates hepatic synthesis of cholesterol and fatty acids, and is a cofactor in many enzymes, including arginase and alkaline phosphatase in the liver. (From AMA Drug Evaluations Annual 1992, p2035)
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D002413	Cations, Divalent	Positively charged atoms, radicals or groups of atoms with a valence of plus 2, which travel to the cathode or negative pole during electrolysis.	Divalent Cations
D003035	Cobalt	A trace element that is a component of vitamin B12. It has the atomic symbol Co, atomic number 27, and atomic weight 58.93. It is used in nuclear weapons, alloys, and pigments. Deficiency in animals leads to anemia; its excess in humans can lead to erythrocytosis.	Cobalt-59,Cobalt 59
D004080	Dihydroorotase	An enzyme that, in the course of pyrimidine biosynthesis, catalyzes ring closure by removal of water from N-carbamoylaspartate to yield dihydro-orotic acid. EC 3.5.2.3.	Carbamoylaspartic Dehydrase,Dihydro-Orotase,Dihydro-Orotate Amidohydrolase,Amidohydrolase, Dihydro-Orotate,Dehydrase, Carbamoylaspartic,Dihydro Orotase,Dihydro Orotate Amidohydrolase
D004926	Escherichia coli	A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc.	Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D005033	Ethylmaleimide	A sulfhydryl reagent that is widely used in experimental biochemical studies.	N-Ethylmaleimide,N Ethylmaleimide
D000595	Amino Acid Sequence	The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION.	Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D001059	Apoproteins	The protein components of a number of complexes, such as enzymes (APOENZYMES), ferritin (APOFERRITINS), or lipoproteins (APOLIPOPROTEINS).	Apoprotein